Enterostatin
Enterostatin is the aminoterminal pentapeptide of procolipase that is released by proteolytic activity when procolipase is converted into colipase (9). The procolipase gene is expressed in the exocrine pancreas and the gastric and duodenal mucosa (25, 34, 53). In the gastric mucosa, the gene appears to be concentrated in enterochromaffin cells. More recently, procolipase and enterostatin were shown to be present in specific brain regions including the amygdala and hypothalamus (12).
Enterostatin Effects on Feeding Behavior. The peptide enterostatin has a dose-dependent and selective effect to inhibit fat intake in a number of dietary paradigms. The first criteria for establishing the physiological role of a peptide on feeding behavior is that it inhibits food or macronutrient intake in rats adapted to a three-choice macronutrient diet of fat, carbohydrate and protein (7, 36, 37). Enterostatin reduced intake of the fat macronutrient, but had no effect on either carbohydrate or protein intake. In a two-choice high-fat (HF) and low-fat (LF) diet paradigm experiment, enterostatin reduced only intake of the HF diet, but not of the LF diet (15) Similarly, enterostatin reduced intake of a single dietary source when the source was HF (17), but not when LF. The ability of enterostatin to selectively inhibit fat intake on a two- or three-choice feeding paradigm has been demonstrated after administration of enterostatin by either intraperitoneal, intracerebroventricular (icv), intraduodenal/intragastric, and near celiac arterial injection (15, 16, 19, 22, 27, 29, 52, 57). Similar to other gut peptides, enterostatin appeared to have at least two sites of action, one in the gastrointestinal tract and one in the central nervous system (20, 49, 57).
While the majority of the feeding studies with enterostatin have been performed in overnight fasted rats that have been previously adapted to the experimental diets, the selective effects towards dietary fat have been shown in free-feeding rats injected at the start of the dark cycle. The potency of enterostatin is reflected in the long duration of action on feeding, lasting up to six hours after a single injection in rats adapted to a six-hour feeding schedule, and lasting up to 24 hours after a single injection in rats adapted to ad-libitum feeding. Chronic icy administration of enterostatin from mini-osmotic pumps also attenuated the daily intake of dietary fat in rats fed either a single-choice HF diet or a two-choice HF/LF diet (15, 35). The decrease in daily food intake was accompanied by a reduction in fat deposition and body weight gain. However, in rats chronically treated with enterostatin and fed a low-fat diet for seven days, no significant reduction was seen in either energy intake or change in body weight gain. An intriguing characteristic of the response to enterostatin in both acute and chronic studies was that the reduction in intake of dietary fat is not compensated by an increase in the intake of other macronutrients when a dietary choice is available. This may result from a concomitant increase in corticotropin releasing hormone (CRH) secretion since enterostatin is known to activate the hypothalamic-pituitary-adrenal (HPA) axis (35).
Enterostatin has also been shown to reduce food intake in rabbits, sheep, and baboons (8, 30, 51). However, all of these studies were performed with single-choice diets. In humans, enterostatin administered by intravenous injection was found to reduce the subjective feeling of hunger (44), although was not found to reduce food intake (43).
Enterostatin effects on fat intake appear to be expressed at both gastrointestinal and central nervous system (CNS) sites. The response to peripherally-administered enterostatin was found to be mediated through the hepatic vagus nerve; the response was abolished by either selective hepatic vagotomy or capsaicin treatment (32, 49). Within the CNS, enterostatin was found to act on both the amygdala and paraventricular nucleus (PVN) (12, 14, 20). Enterostatin inhibited fat intake by way of a pathway that contained both serotonergic (55) and kappa-opioidergic (38) neurons. Kappa-opioidergic agonists inhibited the enterostatin effects on feeding, and a K-opioidergic antagonist or nor-Binaltorphamine (BNI) mimicked the effect of enterostatin on selective fat intake (1, 38). In contrast, the general serotonergic antagonist, metergoline but not a 5HT2 receptor antagonist, blocked the response to icy-administered enterostatin (57), and serotonin injections into the PVN inhibited dietary fat intake (10, 45).
A physiological regulator of feeding behavior must be effective at dose levels that are present in the animal. The in vivo concentration of enterostatin has not been established, due to problems in measuring enterostatin. Antibodies that are selective to enterostatin that could be used to analyze tissue levels of enterostatin have been difficult to find. The current values for enterostatin all appear very high, for example, plasma serum enterostatin of 5-40 nM in humans (4) and rats, cerebral spinal fluid enterostatin of 18-92 ng/ml, and brain enterostatin levels of 2.5 nmoles/g tissue (11). A suggestion of the existence of multiple forms of enterostatin in rats and in humans because of genetic polymorphisms in the enterostatin region of the procolipase parent molecule further complicates the efforts to measure enterostatin (11, 46). However, other data have disputed the suggestion of multiple forms (53, 54). Despite these measurement problems, enterostatin-like immunoreactivity has been shown to increase both in human serum and urine after a meal in a biphasic manner (4), and in lymph fluid of cats (50) and serum of rats after feeding (9).
Enterostatin regulation of insulin secretion. Several studies have shown that enterostatin inhibits insulin secretion (24, 26, 28, 39, 42, 47). In vivo perfusion of isolated islets and of the rat pancreas has been used to demonstrate that enterostatin directly inhibits insulin release from islet cells induced by either glucose, tolbutamide, or arginine. (39) Enterostatin (10−9 to 10−5 M) inhibited insulin secretion from islets incubated in the presence of 16.7 mM glucose in a dose-dependent manner. Enterostatin also inhibited insulin secretion stimulated by glybenclamide (5.0 and 10 μM), phorbol 12-myristate-13-acetate (TPA) (50 and 100 nM), and the kappa-opioid agonist U50,488 (100 nM). The inhibitory effect of enterostatin on TPA-induced insulin secretion was attenuated, but still remained in the absence of extracellular Ca2+. The enterostatin inhibition of insulin secretion was blocked by 8-Br-cAMP (1 mM), independent of extracellular Ca2+. Enterostatin reduced the increase in intracellular cyclic AMP content produced by U50,488 (100 nM), in a manner parallel with changes in insulin release (42).
In vivo studies also have shown a reduction in insulin levels without any changes in plasma glucose suggesting an improvement in insulin sensitivity (15, 35). This occurred after both peripheral and central administration of enterostatin, reflecting both direct effects on the islet cells and indirect effects by way of a reduction in vagal stimulation to the pancreas.
Other Effects of Enterostatin. Enterostatin also been shown to affect gastrointestinal motility and gastric emptying (21, 40). The inhibition of gastric emptying was observed only after intracerebroventricular administration of enterostatin, but not after either intraperitoneal or intragastric administration, suggesting that enterostatin also affects efferent vagal activity. However, the inhibitory effect of enterostatin on consumption of a high fat diet was not related to the slowdown of gastric emptying (21). Enterostatin also had direct effects on pig intestine to prolong the quiescent phase I period of peristalsis, which slows down the absorption of nutrients and prolongs intestinal transit time. Enterostatin may also reduce cholesterol levels (48).
Enterostatin also has shown a number of autonomic and endocrine effects in addition to the effect on insulin secretion. It enhanced corticosterone secretion (35) and sympathetic stimulation to brown adipose tissue (32, 33), which would increase thermogenesis (41). These responses, in addition to the suppression of dietary fat intake, help explain the reduction in weight gain and body fat that was seen in rats treated chronically with either peripheral or central enterostatin (15, 35).
Circulating enterostatin. Enterostatin absorption across the intestine was found to be limited and slow, occurring mainly into lymphatic system. Detailed information of the changes in plasma enterostatin or brain uptake of enterostatin after a meal currently exist that would allow a temporal comparison with the termination of feeding and the development of satiety. The data that are available indicate the rise in plasma immunoreactive-like enterostatin activity is slow and does not peak until at least 60 minutes after feeding, which is inconsistent with a theory that an increase in circulating enterostatin plays a role in the termination of the immediate meal.
The presence of procolipase mRNA in the CNS together with enterostatin-like immunoreactivity has been demonstrated (12). Enterostatin also was found at high levels in the cerebrospinal fluid of rats. A hypothesis that this central system is important in determining the appetite for dietary fat is consistent with the evidence that endogenous production of enterostatin is reciprocally related to voluntary selection of fat across and within rat strains.
Enterostatin Receptors. Based on the areas responding to enterostatin, receptors would be expected to be located in brain, pancreas, and the gastrointestinal tract. Enterostatin has been shown not to bind to the galanin or Neuropeptide Y1 receptors (17), kappa-opioid receptors or cholecystokinin A receptors (13) Low affinity enterostatin binding was shown to a brain membrane preparation (Kd 230 nM) (56) and to SK-N-MC neuroepithelioma cells (Kd 40 nM) (2). The dose-response curve to enterostatin is biphasic, exhibiting an inhibition of food intake at lower doses, but stimulation of food intake at higher doses (22). However, since enterostatin has been shown to be biologically active on food intake at extremely low doses compared to other peptides and to inhibit insulin secretion from isolated pancreatic islets at doses of 10−10 to 10−6M, a proposed low affinity casomorphin binding site probably is not the biologically important enterostatin receptor that inhibits fat intake and insulin secretion. More recently the beta subunit of ATP synthase has been identified as an enterostatin receptor. The initial suggestion came from affinity chromatography studies (59). Subsequently, it has been shown that enterostatin and its antagonist beta-casomorphin bind to this purified protein, that binding can be associated with biological activity across a range of analogs, and that this protein is present in the plasma membranes of tissues that are responsive to enterostatin (amygdala, pancreas, liver). (60)
Angiogenesis
In an adult, two types of blood vessels can potentially be found. The normal blood vessel is a resting, quiescent, fully developed vessel. A second form, a proliferating or developing blood vessel, occurs rarely during the normal life cycle (only in early development and reproduction, e.g., menstrual cycle and pregnancy). In contrast, the process of angiogenesis, the proliferation and development of new blood vessels, often occurs in wound healing and in pathological processes, e.g., tumor growth. Angiogenesis is a complex process involving many stages, including extracellular matrix remodeling, endothelial cell migration and proliferation capillary differentiation, and anastomosis. All detectable solid tumors (tumors over 2 mm in diameter) exploit angiogenesis to supply the needed blood to proliferating tumor cells. Studies have demonstrated that the level of vascularization in a tumor is strongly associated with metastasis in melanoma, breast, and lung carcinomas. See R. Bicknell, “Vascular targeting and the inhibition of angiogenesis,” Annals of Oncology, vol. 5, pp. 45-50 (1994).
Angiogenesis inhibitors have been suggested to intervene into neoplastic processes. See G. Gasparini, “The rationale and future potential of angiogenesis inhibitors in neoplasia,” Drugs, vol. 58, pp. 17-38 (1999). The inhibitory agents block angiogenesis, thereby causing tumor regression in various types of neoplasia. Known therapeutic candidates include naturally occurring angiogenic inhibitors (e.g., angiostatin, endostatin, platelet factor-4, somatostatin and its analogs), specific inhibitors of endothelial cell growth (e.g., TNP-470, thalidomide, interleukin-12), agents that neutralize angiogenic molecules (e.g., antibodies to fibroblast growth factor or vascular endothelial growth factor), suramin and its analogs, tecogalan, agents that neutralize receptors for angiogenic factors, agents that interfere with vascular basement membrane and extracellular matrix (e.g., metalloprotease inhibitors, angiostatic steroids), and anti-adhesion molecules (e.g., antibodies such as anti-integrin alpha v beta 3). See L. Rosen, “Antiangiogenic strategies and agents in clinical trials,” Oncologist, vol. 5, supplement 1, pp. 20-27 (2000).
Abnormal angiogenesis occurs when improper control of angiogenesis causes either excessive or insufficient blood vessel growth. Excessive blood vessel proliferation favors tumor growth and development of distant metastases, blindness, skin disorders such as psoriasis, and rheumatoid arthritis. Diseases or conditions that have been associated with undesired vascularization include, for example, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivits, Vitamin A deficiency, atopic keratitis, contact lens overwear, superior limbic keratitis, pterygium keratitis sicca, Sjogren's syndrome, acne rosacea, phylectenulosis, syphilis, myobacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, trauma, rheumatoid arthritis, systemic lupus, polyarteritis, Wegener's sarcoidosis, scleritis, Stevens-Johnson disease, radial keratotomy, macular degeneration, sickle cell anemia, sarcoidosis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, carotid obstructive disease, chronic uveitis, chronic vitritis, Lyme's disease, Eales' disease, Behcet's disease, myopia, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, post-laser complications, abnormal proliferation of fibrovascular or fibrous tissue, hemangiomas, Osler-Weber-Rendu disease, solid tumors, blood borne tumors, acquired immune deficiency syndrome, ocular neovascular disease, age-related macular degeneration, osteoarthritis, diseases caused by chronic inflammation, Crohn's disease, ulcerative colitis, tumors of rhabdomyosarcoma, tumors of retinoblastoma, tumors of Ewing's sarcoma, tumors of neuroblastoma, tumors of ostteosarcoma, leukemia, psoriasis, atherosclerosis, pemphigoid, infections causing retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, proliferative vitreoretinopathy, Bartonellosis, acoustin neuroma, neurofibroma, trachoma, pyogenic granulomas, obesity, corneal neovascularization, malignant tumor growth beyond 2 mm, benign tumors, benign functional endocrine tumors, arterial/venous malformations, primary hyperparathyroidism, secondary hyperparathyroidism, and tertiary hyperparathyroidism. Other angiogenic-related diseases may include, for example, diseases associated with rubeosis (neovascularization of the angle), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy. Any disease having a known angiogenic counterpart could potentially be treated with an anti-angiogenic factor, e.g., psoriasis. See D. Creamer et al., “Overexpression of the angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase in psoriatic epidermis,” Br. J. Dermatol., vol. 137, pp. 851-855 (1997).
Angiogenesis is a prominent contributor to solid tumor growth and the formation of distant metastases. Several experimental studies have concluded that primary tumor growth, tumor invasiveness, and metastasis all require neovascularization. The process of tumor growth and metastasis is complex, involving interactions among transformed neoplastic cells, resident tissue cells (e.g., fibroblasts, macrophages, and endothelial cells), and recruited circulating cells (e.g., platelets, neutrophils, monocytes, and lymphocytes). A possible mechanism for the maintenance of tumor growth is an imbalance, or disregulation, of stimulatory and inhibitory growth factors in and around the tumor. Disregulation of multiple systems allows the perpetuation of tumor growth and eventual metastasis. Angiogenesis is one of many systems that is disregulated in tumor growth. In the past it has been difficult to distinguish between disregulation of angiogenesis and disregulation of other systems affecting a developing tumor. Another complicating factor is that aggressive human melanomas mimic vasculogenesis by producing channels of patterned networks of interconnected loops of extracellular matrix, in which red blood cells, but not endothelial cells, are detected. See A. J. Maniotis et al., “Vascular channel formation by human melanoma cells in vivo and in vitro: Vasculogenic mimicry,” Am. J. Pathol., vol. 155, pp. 739-52 (1999). These channels may facilitate perfusion of tumors, independent of perfusion from angiogenesis.
A tumor cannot expand beyond approximately 2 mm without a blood supply to provide nutrients and remove cellular wastes. Tumors in which angiogenesis is important include solid tumors, and benign tumors including acoustic neuroma, neurofibroma, trachoma, and pyogenic granulomas. Inhibiting angiogenesis could halt the growth and potentially lead to regression of these tumors. Angiogenic factors have been reported as being associated with several solid tumors, including rhabdomyosarcoma, retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma.
Angiogenesis has also been associated with some non-solid tumors, including blood-born tumors such as leukemia, various acute or chronic neoplastic diseases of the bone marrow marked by unrestrained proliferation of white blood cells, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen. It is believed that angiogenesis may play a role in the abnormalities in the bone marrow that give rise to leukemias and multiple myelomas.
Anti-angiogenic factors inhibit tumor growth beyond 2 mm by inhibiting the angiogenic response and thus inhibiting blood vessel growth to the tumor. Although angiogenesis in a tumor may begin at an early stage, a tumor requires a blood supply to grow much beyond about 2 mm. Up to 2 mm diameter, tumors can survive by obtaining nutrients and oxygen by simple diffusion. Most anti-angiogenic factors are not cytotoxic, i.e., capable of killing the tumor cells directly. Small tumors of a size about 1 mm3 can be effectively inhibited and destroyed by factors, either endogenous or exogenous, that stimulate the immune system. It is generally accepted that once a tumor has reached a critical size, the immunological system is no longer able to effectively destroy the tumor; i.e., there is a negative correlation between tumor size and immune competence. See A. K. Eerola et al., “Tumour infiltrating lymphocytes in relation to tumour angiogenesis, apoptosis,” Lung Cancer, vol. 26, pp. 73-83 (1999); and F. A. Wenger et al., “Tumor size and lymph-node status in pancreatic carcinoma—is there a correlation to the preoperative immune function?,” Langenbecks Archives of Surgery, vol. 384, pp. 473-478 (1999). Early adjuvant use of an effective anti-angiogenic agent to preclude development of tumor metastases beyond 1 to 2 mm3 may allow more effective tumor attack and control by the body's immunological mechanisms. In addition, prolonged adjuvant use of a non-toxic angiogenic inhibitor may prevent tumor dissemination by blocking the growth of vessels required for the transport of tumor cells that would form metastatic foci.
Angiogenesis has also been implicated in obesity. Several mice strains, both young and aged animals, used as obesity models treated with anti-angiogenic agents lost weight. See M. A. Rupnick et al., “Adipose tissue mass can be regulated through the vasculature,” PNAS, vol. 99, pp. 10730-10735 (2002). This same study also found that adipose tissue mass was reduced by the anti-angiogenic compounds.
New anti-angiogenic factors are needed, in particular, compounds that not only inhibit new angiogenic growth, but also that degrade existing capillary networks. Very few anti-angiogenic factors have been reported to diminish existing capillary networks.